Composition comprising recombinant fusion protein of pathogenic antigen protein and flagellin of vibrio vulnificus for preventing, alleviating, or treating aging

ABSTRACT

The present invention relates to a composition for preventing, improving, or treating aging, wherein the composition comprises a recombinant protein of flagellin, which is the constituent of  Vibrio vulnificus  flagella, fused with a pathogenic protein antigen, as an active component. According to the present invention, the recombinant protein of the present invention can improve external and internal aging-related malfunctions and enhance immunity. Also, the composition of the present invention can easily perform immunization through mucosal administration.

TECHNICAL FIELD

The present invention was made with the support of the National ResearchFoundation of Korea, under Project No. 2012-0002472, which was conductedin the program titled “General Researcher Support Project/FemaleScientist Support Project” in the project named “Research for theMechanism of Aging Dependent Innate immune dysfunction” by the ChonnamNational University under the management of the National ResearchFoundation of Korea, from May 1, 2010 to Apr. 30, 2013.

Furthermore, the present invention was made with the support of theMinistry of Knowledge Economy, Republic of Korea, under Project No.RTI-0501-01, which was conducted in the program titled “New Vaccines andImmune Disease Therapeutic Agents Development Project” in the projectnamed “Development of Anti-aging Vaccine Adjuvant” by the ChonnamNational University under the management of the Ministry of KnowledgeEconomy, Republic of Korea, from Jul. 1, 2011 to Jun. 30, 2012.

Furthermore, the present invention was made with the support of theMinistry of Education, Science, and Technology of Republic of Korea,under Project No. 2011-0030034, which was conducted in the programtitled “Overseas Excellent Research Institutions Inducement Project” inthe project named “Age-Related Cellular Function Regulation” by theChonnam National University Hwasun Hospital under the management of theMinistry of Education, Science, and Technology of Republic of Korea,from Jul. 1, 2011 to Jun. 30, 2012.

This application claims priority from Korean Patent Application No.10-2012-0138234 filed on Nov. 30, 2012, the disclosure of which isincorporated herein by reference.

The present invention relates to a composition for preventing,improving, or treating aging, the composition comprising a recombinantprotein of flagellin, which is the constituent of Vibrio vulnificusflagella and an agonist of a toll-like receptor 5 (TLR-5), fused with apathogenic antigen, and to a method for preventing, improving, ortreating aging.

BACKGROUND ART

Humans are facing various problems not seen before due to the advent ofan aging society caused by a prolonged average life span. Insocio-economic aspects, the elderly sustenance allowance per head isexpected to increase due to the increase in the elderly population andthe reduction in the productive age population, and the interest in theimprovement of the quality of life of the elderly is also a growingtrend. As the social demands for the healthy and happy life of theelderly increase as described above, studies on the change inaging-related disease aspect and prevention of aging-related diseasesare under active progress.

The aging process causes a wide variety of changes. Various changesappear, including reduced functions of respective main tissues, foodintake and digestive disorders, reduced brain functions includingdefective memory, and reduced cardiovascular functions, as well asvarious appearance changes, such as skin wrinkles, hair decoloration,spine curving, and the change in motion. Moreover, these changes inducethe reduction of functions and diseases of the respective tissues, andtherefore, it is very important to understand causes of the reduction ofexternal and internal functions due to aging and develop techniques ofregulating the functions.

Besides, one of the large changes in functions due to aging is thereduction in immune function. This is called immunosenescence. Whenpathogens invade a host, the host defection action is made by two immunesystems, an innate immune system and an adaptive immune system. Ofthese, the innate immune system is activated immediately afterinfection, to promptly regulate infecting pathogens, and takes charge ofthe initial infection until the adaptive immune system is activated. Inthis innate immune system, a receptor recognizing “pathogen associatedmolecular patterns (PAMPs) existing in the pathogens is called “patternrecognition receptors (PRRs), and these receptors are called toll-likereceptors in a mammal. So far, 13 kinds of TLRs have been found, andstudies on agonists of the respective TLRs have been actively conducted(Shizuo Akira et al, Cell, Pathogen Recognition and Innate Immunity,124(4):783-801, 2006). PRRs like TLRs exist on the cell surface or inthe protoplasm, and have been known to regulate the innate immuneresponse by various stimuli of PAMPs, and further regulate the adaptiveimmune response. Therefore, the agonists of TLRs may be target materialssuitable for the development of various immunomodulators and vaccineadjuvants.

Of studies on aging, the fields that have received the most attention sofar are the life span adjustment of aging or functional recovery ofaging. Recently, studies on the extension of life span are rapidlyincreasing through various methods, such as by inhibiting the expressionof a particular gene or overexpressing the particular gene in studiesusing drosophila models or nematodes, restricting diet, or treating withrapamycin. In addition, the interest in the maintenance of functions orrecovery of functions, instead of the simple extension of life span, isalso a growing trend. However, the regulation of a particular genereferring to the results shown in lower animal models may cause otherfunctional side effects, and thus has a limitation in the application tohumans. Moreover, the treatment with a drug, such as rapamycin, maygreatly influence the immune function.

Throughout this application, various patents and publications arereferenced, and citations are provided in parentheses. The disclosure ofthese patents and publications in their entities are hereby incorporatedby references into this application in order to more fully describe thisinvention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THE INVENTION Technical Problem

The present inventors have endeavored to develop a material capable ofpreventing, improving, or treating dysfunction due to aging. As aresult, the present inventors have confirmed that the expressions ofmost toll-like receptors are reduced but toll-like receptor-5 is wellexpressed to keep functions thereof in aging immune cells, and confirmedthat, when aged mice are immunized with a recombinant protein offlagellin, which is the constituent of Vibrio vulnificus flagella, fusedwith a pathogenic protein antigen of a pathogen, the immunity of theaged mice is activated and external and internal functions of the agedmice are improved, and thus have completed the present invention.

Therefore, an object of the present invention is to provide acomposition for preventing, improving, or treating aging, thecomposition comprising, as an active ingredient, a recombinant proteinof flagellin, which is the constituent of Vibrio vulnificus flagella,fused with a pathogenic protein antigen.

Another object of the present invention is to provide a composition forpreventing, improving, or treating hair-related disease, the compositioncomprising the recombinant protein as an active ingredient.

Still another object of the present invention is to provide acomposition for preventing, improving, or treating eye-related disease,the composition comprising the recombinant protein as an activeingredient.

Still another object of the present invention is to provide acomposition for preventing, improving, or treating bowel disease, thecomposition comprising the recombinant protein as an active ingredient.

Still another object of the present invention is to provide acomposition for preventing, improving, or treating bone disease, thecomposition comprising the recombinant protein as an active ingredient.

Another object of the present invention is to provide a method forpreventing, improving, or treating aging.

Other objects and advantages of the present invention will becomeapparent from the detailed description to follow taken in conjugationwith the appended claims and drawings.

Technical Solution

According to an aspect of the present invention, the present inventionprovides a composition for preventing, improving, or treating aging, thecomposition comprising, as an active ingredient, a recombinant proteinof flagellin, which is the constituent of Vibrio vulnificus flagella,fused with a pathogenic protein antigen.

The present inventors have endeavored to develop a material capable ofpreventing, improving, or treating dysfunction due to aging. As aresult, the present inventors have confirmed that the expressions ofmost toll-like receptors are reduced but toll-like receptor-5 is wellexpressed to keep functions thereof in aging immune cells, and confirmedthat, when aged mice are immunized with a recombinant protein offlagellin, which is the constituent of Vibrio vulnificus flagella, fusedwith a pathogenic protein antigen, the immunity of the aged mice isactivated and external and internal functions of the aged mice areimproved.

The largest feature of the present invention is to use a recombinantprotein of flagellin, which is the constituent of Vibrio vulnificusflagella, fused with a pathogenic protein antigen.

As used herein, the term “flagellin” refers to a unit moleculeconstituting flagella, which are the cilia to determine the mobility ofbacteria. The flagellin of the present invention includes all flagellinsthat act as a TLR-5 activator of Vibrio vulnificus. According to anembodiment of the present invention, the flagellin of the presentinvention is FlaB, which is the flagellin of Vibrio vulnificus.

Examples of the pathogenic protein antigen usable herein may include anα-helix domain of surface protein A (PspA) and pneumococcal surfaceprotein A (PsaA) of Streptococcus pneumonia; subunit hemagglutinin (HA)and neuraminidase (NA) of influenza virus; and spike (S) protein ofsevere acute respiratory syndrome virus (SARS virus), but are notlimited thereto. According to an embodiment of the present invention,the pathogenic protein antigen is surface protein A (PspA) ofStreptococcus pneumonia.

According to an embodiment of the present invention, the recombinantprotein of the present invention is FlaB-PspA protein having an aminoacid sequence of SEQ ID NO: 1.

As used herein, the term “aging” refers to a functional, structural, andbiochemical procedure that continuously occurs in a subject from birthuntil death. The aging occurs in the overall cells and body tissuesconstituting the subject, and indicates the decrease in the metabolicrate, the increase in diseases, and the deterioration in adaptability,ultimately leading to death in cells and the whole body. For example,examples of aging include external changes, such as the increase in skinwrinkles, the reduction of hair gloss, hair decoloration, hair loss, thethickness reduction of the dermal layer in which hair follicles arepresent, the reduction in the number of follicles, spine curving, andthe reduction in exercising; internal changes, such as the reduction inimmune functions and the reduction in functions of main tissues; and theconsequent occurrence of diseases of respective tissues, but are notlimited thereto.

According to an embodiment of the present invention, the recombinantprotein of the present invention enhances immunity. As used herein, theterm “enhancing immunity” refers to keeping the immune response oractivity of the in vivo immune system at a level of a non-aged controlgroup or enhancing the same to a level of an aged control group.According to another embodiment of the present invention, therecombinant protein of the present invention prevents the reduction offunctionality of immune-related organs due to aging or enhances thefunctionality compared with the aged control group, by increasing theproduction of secretory globulin A (secretory lgA, slgA) antibody,increasing the frequency of hematopoietic stem cells which areessentially associated with T cell differentiation, preventing thymicinvolution, or preventing the hypertrophy of mesenteric lymph nodes(MLNs) or spleens.

According to an embodiment of the present invention, the recombinantprotein of the present invention prevents, improves, or treats thedeteriorations in metabolic functions, functions of skin tissues,functions of intestinal tissues, functions of muscular tissues, brainfunctions, or cardiovascular functions. According to another embodimentof the present invention, the recombinant protein of the presentinvention prevents, improves, or treats the deteriorations in metabolicfunctions, functions of skin tissues, functions of intestinal tissues,or functions of muscular tissues. According to a particular embodimentof the present invention, the recombinant protein of the presentinvention prevents, improves, or treats the deteriorations in functionsof the dermal tissue in which hair follicles are present, functions oflarge intestine tissues, or functions of muscular tissues.

According to another embodiment of the present invention, the presentinvention provides a composition for preventing, improving, or treatingmetabolic disease, the composition comprising the recombinant protein asan active ingredient.

As used herein, the term “metabolic disease” refers to a syndrome inwhich risk factors, such as obesity, diabetes, hypertriglyceridemia,hypertension, cardiovascular disease, and blood clotting, are showntogether. The syndrome per se is not fatal, but has a predisposition tosevere diseases, such as diabetes or ischemic cardiovascular diseases,resulting in a great threat to modern people. The syndrome was oncecalled several names, including syndrome X, since the cause of thesyndrome was not known, but recently, the syndrome was officially namedmetabolic syndrome or insulin resistance syndrome through adulttreatment program III (ATP III) established by the World HealthOrganization and the US National Institutes of Health. According to anembodiment of the present invention, the metabolic disease of thepresent invention is obesity.

According to another embodiment of the present invention, the presentinvention provides a composition for preventing, improving, or treatinghair-related disease, the composition comprising the recombinant proteinas an active ingredient.

Examples of the hair-related disease which can be prevented, improved,or treated by the composition of the present invention include thereduction of hair gloss, hair decoloration, hair loss, the thicknessreduction of the dermal layer in which hair follicles are present, thereduction in the number of follicles, and the like, but are not limitedthereto.

According to still another embodiment of the present invention, thepresent invention provides a composition for preventing, improving, ortreating bowel disease, the composition comprising the recombinantprotein as an active ingredient.

Examples of the bowel disease which can be prevented, improved, ortreated by the composition of the present invention include irritablebowel syndrome (IBS), uncontrolled diarrhea-associated irritable bowelsyndrome (dIBS), Crohn's disease, traveler's diarrhea, ulcerativecolitis, enteritis, small intestine bacterial overgrowth, chronicpancreatitis, pancreatic insufficiency, colitis, diverticular disease,hepatic encephalopathy, and hernia, but are not limited thereto.According to an embodiment of the present invention, the bowel diseaseof the present invention is enteritis or hernia. According to anotherembodiment of the present invention, the bowel disease of the presentinvention is colitis or hernia.

According to still another embodiment of the present invention, thepresent invention provides a composition for preventing, improving, ortreating bone disease, the composition comprising the recombinantprotein as an active ingredient.

Examples of the bone disease which can be prevented or treated by thecomposition of the present invention include osteoporosis, scoliosis,osteomalacia, rickets, bone metastasis of cancer cells, bone damagecaused by bone metastases of cancer cells, osteolysis caused by bonemetastases of cancer cells, fibrous dysplasia, aplastic bone disease,metabolic bone disease, rheumatoid arthritis, osteoarthritis,degenerative arthritis, and disc disease, but are not limited thereto.According to an embodiment of the present invention, the bone-relateddisease of the present invention is osteomalacia, metabolic bonedisease, rheumatoid arthritis, osteoarthritis, or degenerativearthritis. According to another embodiment of the present invention, thebone disease of the present invention is osteoporosis or osteomalacia.

The composition for preventing, improving, or treating the foregoingdiseases of the present invention contains the foregoing recombinantprotein as an active ingredient, and thus descriptions of overlappingcontents with the recombinant protein are omitted to avoid excessivecomplication of the specification due to repetitive descriptionsthereof.

The composition of the present invention may be provided as apharmaceutical composition.

According to an embodiment of the present invention, the composition ofthe present invention is a pharmaceutical composition comprising (a) apharmaceutically effective amount of the recombinant protein any one ofthe recombinant proteins of the present invention; and (b) apharmaceutically acceptable carrier. The term “pharmaceuticallyeffective amount” refers to an amount enough to show and accomplishefficacies and activities of the recombinant protein of this inventionas described above.

According to the present invention, the pharmaceutical composition maycontain pharmaceutically acceptable carriers. The pharmaceuticallyacceptable carrier may be conventional one for formulation, includinglactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable,potassium phosphate, arginate, gelatin, potassium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils, but not limitedto. The pharmaceutical composition according to the present inventionmay further include a lubricant, a humectant, a sweetener, a flavoringagent, an emulsifier, a suspending agent, and a preservative. Details ofsuitable pharmaceutically acceptable carriers and formulations can befound in Remington's Pharmaceutical Sciences (19th ed., 1995), which isincorporated herein by reference.

The pharmaceutical composition of this invention may be administeredmucosally, orally, or parenterally, according to an embodiment,mucosally injection.

A suitable dosage amount of the pharmaceutical composition of thepresent invention may vary depending on pharmaceutical formulationmethods, administration methods, the patient's age, body weight, sex,pathogenic state, diet, administration time, administration route, anexcretion rate and sensitivity for a used pharmaceutical composition,and physicians of ordinary skill in the art can determine an effectiveamount of the pharmaceutical composition for desired treatment.

Generally, suitable dosage unit for human host is to administer with thepharmaceutical composition in 0.001-100 mg/kg (body weight).

According to the conventional techniques known to those skilled in theart, the pharmaceutical composition may be formulated withpharmaceutically acceptable carrier and/or vehicle as described above,finally providing several forms including a unit dose form and amulti-dose form. Formulation may be oil or aqueous media, resuspensionor emulsion, extract, powder, granule, tablet and capsule and furthercomprise dispersant or stabilizer.

The composition of the present invention may be provided as a foodcomposition.

According to an embodiment of the present invention, the composition ofthe present invention is a food composition comprising (a) asitologically effective amount of the recombinant protein any one of therecombinant proteins of the present invention; and (b) a sitologicallyacceptable carrier. When the composition of the present disclosure isprepared as a food composition, the food composition of the presentdisclosure may comprise, in addition to the recombinant protein of thepresent disclosure as active ingredients, ingredients commonly added forpreparation of food. For example, proteins, carbohydrates, fats,nutrients, seasoning or flavors may be added. The carbohydrate may be,for example, a sugar such as a monosaccharide, e.g. glucose, fructose,etc., a disaccharide, e.g. maltose, sucrose, oligosaccharide, etc. or apolysaccharide, e.g. dextrin, cyclodextrin, etc. or a sugar alcohol suchas xylitol, sorbitol, erythritol, etc. The flavor may be a naturalflavor [thaumatin, stevia extract (e.g. rebaudioside A, glycyrrhizin,etc.]) or a synthetic flavor (saccharin, aspartame, etc.). For example,when the food composition of the present disclosure is prepared as adrink, it may further comprise, in addition to the recombinant proteinof the present disclosure as the active ingredient, citric acid,high-fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruitjuice, eucommia extract, jujube extract, licorice extract, or the like.

The composition of the present invention may be provided as a cosmeticcomposition.

In cases where the composition of the present invention is used toprepare a cosmetic composition, the composition of the present inventioncontains not only the foregoing recombinant protein but also ingredientsnormally used in the cosmetic composition, for example, a carrier andnormally additives, such as an antioxidant, a stabilizer, a solubilizer,vitamins, a pigment, and a flavor.

As the carrier, purified water, monohydric alcohol (ethanol or isopropylalcohol), polyhydric alcohol (glycerol, 1,3-butylene glycol or propyleneglycol), higher fatty acid (palmitic acid or linolenic acid), oil (wheatgerm oil, camellia oil, jojoba oil, olive oil, squalene, sunflower oil,macadamia peanut oil, avocado oil, soybean water-added lecithin or fattyacid glyceride) or the like may be used, but the carrier is not limitedthereto. In addition, a surfactant, a sterilizer, an antioxidant, a UVabsorber, an anti-inflammatory agent, and a refreshing agent may beadded as needed.

The surfactant may include one selected from the group consisting ofpolyoxyethylene, hardened castor oil, polyoxyethylene, oleyl ether,polyoxyethylene monooleate, glyceryl monostearate, sorbitanmonostearate, polyoxyethyelene monostearate, sorbitan, sucrose fattyacid ester, hexaglycerine monolaurate, polyoxyethylene reduced lanolin,POE, glyceryl pyroglutamate, isostearic acid, diester,N-acetylglutamine, and isostearyl ester.

The sterilizer may include one selected from the group consisting ofhinoki thiol, triclosan, chlorhexidine gluconate, phenoxy ethanol,resorcin, isopropyl methyl phenol, azulene, salicylic acid, and zincpyrithione.

As the antioxidant, any one of butyl hydroxyanisole, gallic acid, propylgallate, and erythorbic acid may be used.

As the UV absorbent, any one of benzophenones such as dihydroxybenzophenone, melanin, para-amino benzoic acid ethyl, para-dimethylaminobenzoic acid 2-ethylhexyl ester, cynocite, para-methoxy cinnamic acid2-ethylhexylester, 2-(2-hydroxy-5-methylphenyl)benzotriazole, urocanicacid, and metal oxide microparticles may be used.

For the anti-inflammatory agent, glycyrrhetinic acid dipotassium orallantoin may be used, and as the refreshing agent, capsicum tincture or1-menthol may be used.

The dosage form of the composition is any dosage form that can blend therecombinant protein as an active ingredient, and examples of the dosageform of cosmetics for preventing hair loss may include various forms ofa sol, a gel, an emulsion, oil, wax, aerosol, and the like, such as hairtonic, hair cream, hair lotion, hair shampoo, hair rinse, hairconditioner, hair spray, hair aerosol, pomade, a powder, and a gel, butare not limited thereto.

In still another aspect of this invention, there is provided a methodfor preventing, improving, or treating aging, comprising administeringto a subject in need thereof the composition of the present invention.

Advantageous Effects

The features and advantages of this invention will be summarized asfollows:

(a) The present invention provides a composition for preventing,improving, or treating aging, the composition comprising, as an activeingredient, a recombinant protein of flagellin, which is the constituentof Vibrio vulnificus flagella, fused with a pathogenic protein antigen.

(b) The present invention provides a composition for preventing,improving, or treating metabolic disease, hair-related disease, eyedisease, bowel disease, or bone disease, the composition comprising therecombinant protein as an active ingredient.

(c) The present invention can improve external and internalaging-related malfunctions and enhance immunity by using the recombinantprotein of the present invention.

(d) The present invention can easily perform immunization throughmucosal administration of the composition of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic view with respect to an experiment schedule andan experiment scheme for immunizing aged mice.

FIG. 2 shows graphs illustrating measurement results of changes in foodintake and body weight in aged mice according to the immunization withFlaB-PspA recombinant protein.

FIG. 3 shows morphological change results of aged mice throughcontinuous immunization of an antigen.

FIG. 4 is an IgA reaction graph in aged mice by continuous immunization.

FIG. 5 illustrates histopathological findings with respect to thecutaneous histological change of aged mice by immunization withFlaB-PspA recombinant protein.

FIG. 6 shows the changes of hematopoietic stem cells in marrow cells ofaged mice by immunization with FlaB-PspA recombinant protein.

FIG. 7 shows organs illustrating lymphatic system changes of aged miceby immunization with FlaB-PspA recombinant protein.

FIG. 8 shows the change in bone mineral density of aged mice byimmunization with FlaB-PspA recombinant protein.

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail withreference to examples. These examples are only for illustrating thepresent invention more specifically, and it will be apparent to thoseskilled in the art that the scope of the present invention is notlimited by these examples.

EXAMPLES Materials and Methods

Aged mice (C57BL/6J, aged at least 23 months) were intranasallyimmunized with purified protein and recombinant protein continuously attwo-week intervals. PspA protein and FlaB-PspA protein were purchasedfrom the laboratory of professor Jun-haeng, Lee of the Clinical VaccineDevelopment Project Group of Chonnam National University.

All immunization experiments were conducted in the specific pathogenfree (SPF) facility. Young mice (8-10 week old) were used for a controlgroup. A non-immunized aged mouse group, an aged mouse groupintranasally immunized with PBS (16 μl/mouse) 8 times or more at 2-weekintervals, an aged group intranasally immunized with only PspA (2.5μg/16 μl/mouse) protein 8 times or more at 2-week intervals, and an agedgroup intranasally immunized with FlaB-PspA (6.5 μg/16 μl/mouse)recombinant protein 8 times or more at 2-week intervals were used forimmunization experimental groups.

The morphological and behavioral changes of the aged mice were observedduring the continuous immunization.

Following the immunization, the body weight and feed intake of eachmouse were measured every week. In order to accurately measure thechanges according to the immunization, the mice were separately managedone by one. In addition, 50 g of feed was provided for each mouse, andthen the remainder of the feed was accurately measured at one-weekintervals. Also, the body weight of each mouse was accurately measuredusing an animal scale every week.

In order to compare morphological changes of aged mice according to theimmunization, the appearances of the mice, that is, the hair condition,hair loss, and decoloration was observed and the anus or eyes were alsocontinuously observed, thereby collecting changed patterns.

In order to compare the behavior changes of the aged mice according tothe immunization, the behavioral changes of the mice were observed at acertain point during the immunization. In order to verify the behavioralability of the mice, the aged mice of each group were placed in aconfined space, and the motions of the mice were observed for a periodof time. The motions were compared based on general standard items ofmouse behavioral ability, that is, the motion, the number of times ofstanding on hind legs, the number of times of supporting using forelegs,and the number of times of touching the nose.

During the continuous immunization, in order to verify the change of theimmune response depending on the number of times of immunization, bloodand feces were collected from some mice, and then the change in antibodyproduction was measured using enzyme-linked immunosorbent assay (ELISA).

After immunization for an appropriate period of time, several sampleswere collected from the overall mice, and then the entire changes of theaged mice according to the immunization were measured through variousexperiments.

In order to verify the change of the immune response according to thecontinuous immunization, serum and several mucous samples (feces,saliva, vaginal washing, etc.) were collected from the mice, and inorder to compare the mucosal immune response, the change in secretoryimmunoglobulin A (secretory IgA, slgA) production was measured by ELISA.

In order to compare the morphological changes of the aged mice accordingto the immunization, various tissues including the skin tissue werecollected, and fixed with formalin prior to making a paraffin-block, andpathological findings were compared through Hematoxylin and Eosin (H&E)staining. In addition, with reference to the effect of immunization andthe feed intake through the mucosal immune response, pathologicalfindings of the tissues of small and large intestines, which largelyaccount for the mucosal immunity, were compared and observed through H&Estaining.

In order to compare the antigen-mediated cellular immune response withrespect to lymphocytes separated from immune-related tissues, thelymphocytes were separated from the cervical lymph nodes and spleen andthen the antigen-mediated cellular response was compared by ELISA. Inaddition, in order to compare and observe changed patterns of variousbone marrow cells with respect to the cells separated from the bonemarrow, the bone marrow cells were separated from hind legs of the agedmice, and the comparison was conducted using flow cytometry (FACS,Beckman Coulter). Particularly, the frequency of hematopoietic stemcells essentially associated with T cell differentiation was comparedand observed through immuno-staining using CD34, which is ahematopoietic stem cell indicator.

In order to compare the change in bone mineral density (BMD) of the agedmice according to the immunization with respect to spines separated fromthe mice, the spines were extracted from the mice, and then the bonemineral density (BMD) according to the continuous immunization andkyphosis according to aging of the aged mice were compared and observedusing micro-computer tomography (microCT: Skyscan 1172, Micro PhotonicsInc., US).

Next, for the ongoing study, the morphological and behavioral changes ofthe aged mice according to the continuous immunization were compared andcompared in connection with metabolism. For this, after the blood wascollected from the aged mice, the serum or plasma was separatedtherefrom, and then the changes in hormone-related andmetabolism-related genes were compared. In addition, in order to compareand observe the change in mucosal immunity according to theimmunization, the intestine-associated microenvironment was comparedusing normal microbiota. In addition, the blood and feces were collectedfrom the aged mice continuously immunized with antigens, and then thegene expression pattern was compared and observed through advancedanalysis methods, such as microarray.

Results Example 1: Changes in Feed Intake and Body Weight in Aged MiceImmunized with Flagellin-PspA Recombinant Protein

While the aged mice were intranasally immunized eight times at two-weekintervals, the body weight and the feed intake of the mice of each groupwere measured. The non-immunized aged mice were used as a control group.

The aged mice were intranasally immunized eight times with phosphatebuffered saline (PBS), prepared 2.5 μg of PspA, and 6.5 μg of FlaB-PspArecombinant protein at two-week intervals. The body weight and the feedintake of the mice of each group were measured every week. Themeasurement results are shown in FIGS. 2a and 2 b.

As a result of body weight measurement, it was verified that thenon-immunized mouse group, the group immunized with PBS, the groupimmunized with PspA alone, and the group immunized with FlaB-PspArecombinant protein showed no change in body weight due to theimmunization (FIG. 2a ).

As a result of comparing the feed intake of the aged mice according tothe immunization, the non-immunized mouse group showed no great changein the feed intake over time, and the group immunized with PBS alsoshowed no great change in the feed intake, regardless of eight times ofimmunization at two-week intervals. On the other hand, the groupimmunized with PspA alone was verified to show a gradual increase in thefeed intake during the continuous immunization. Particularly, it wasshown that the group immunized with FlaB-PspA recombinant protein showedan increase in the feed intake through the continuous immunization, andhere, the rate in increase of the feed intake was significantly higherthan that of the group immunized with PspA alone (FIG. 2b ).

As can be seen from the results of example 1, it can be confirmed that,when the aged mice are continuously immunized with antigens, the bodychange is not greatly changed, but the feed intake is greatly increased.Particularly, it can be seen that the immunization with a recombinantprotein including a vaccine adjuvant fused with a pathogenic antigensignificantly increases the feed intake.

Example 2: Observation of Morphological Changes of Mice ThroughContinuous Immunization with Antigen

While the aged mice were intranasally immunized eight times at two-weekintervals, the morphological changes of the mice according to theimmunization were monitored every week. The results are shown in FIG. 3.The non-immunized aged mice were used as a control group.

The aged mice prior to the immunization had no abnormal findings byappearances. There were no abnormal findings in view of hair condition,hair luster, hair decoloration, hair loss, the anus (colitis or hernia),or the eyes (cataracts).

However, as a result of observing the aged mice following theimmunization, it was verified that the non-immunized aged mouse grouphad a worse hair condition than the group immunized eight times attwo-week intervals, and the appearances became generally worse, such asa severe progression of hair decoloration or hair loss. Besides, normalfindings, such as hernia, were severely shown in the anus, and abnormalfindings, such as suspected cataracts, were severely shown also in botheyes (O-control). The group immunized with PBS also had a bad haircondition, and the appearances thereof became severely worse, such assevere progressions of hair decoloration and hair loss. In addition,findings of slight colitis or hernia were shown in the anus, andfindings of suspected cataracts were severely shown in one eye (O-PBS).

On the other hand, as a result of eight times of immunization with PspA,slight decoloration was shown but the hair loss was not severe in theappearance of the aged mice, and abnormal findings were not observed inthe anus or eyes (O-PspA). Particularly, it was verified that, the groupimmunized with FlaB-PspA recombinant protein had a good hair conditionso that the hair condition of the aged mice of the group was verysimilar to that of the aged mice prior to the immunization, and abnormalfindings were observed in neither the anus nor eyes (O-FlaB-PspA).

As can be seen from the results of example 2, it can be verified thatthe appearances of the aged mice are better through the continuousimmunization. It is general that the aging causes a severe progressionof hair decoloration or hair loss and abnormal findings, such ascataracts occurring in eyes. Rodents showed abnormal findings, such ashernia, in the anus due to the reduction in the muscle amount. However,the continuous immunization with antigens could be verified to preventthe occurrence of such abnormal findings. Particularly, it can be seenthat, as for the group immunized with a recombinant protein including avaccine adjuvant fused with a pathogenic antigen, the appearances of theaged mice are maintained over time or have a better condition.

Example 3: Change in IgA Reaction of Aged Mice Through ContinuousImmunization with Antigen

During the continuous immunization, the feces were collected from themice of each group after each immunization to verify the IgA reaction ofthe aged mice according to the immunization by ELISA. The results areshown in FIG. 4. The non-immunized aged mice were used as a controlgroup.

The non-immunized aged mice had no great difference in the IgA responseduring the immunization (0-control). It was verified that the groupimmunized with PBS alone showed no great difference in the IgA response,and then showed a slight increase in the IgA response after the sixthimmunization, but there is no great increase in the IgA response(O-PBS). The group treated with PspA alone showed the IgA response afterthe fourth immunization, but showed no great difference after that(O-PspA). Whereas, as for the group immunized with FlaB-PspA recombinantprotein, the IgA response was significantly increased depending on thenumber of times of immunization (O-FlaB-PspA).

As can be seen from the results of example 3, it was verified that, as aresult of verifying the IgA reaction with respect to the mucosal immunereaction of the aged mice through the continuous immunization, the IgAreaction was significantly increased depending on the immunization inthe group immunized with FlaB-PspA recombinant protein while no greatchange in the IgA reaction or a slight IgA reaction was shown in theother groups. Judging from the results, the mucosal immunity of the agedmice is activated by the recombinant protein.

Example 4: Skin Histological Change of Aged Mice Through Immunizationwith Recombinant Protein

The results of example 2 indicated that the hair condition of the agedmice became very favorable in the group continuously immunized withantigens, particularly, the recombinant protein. In order to prove theseresults in more detail, the results were confirmed through H&E staining.The H&E staining results are shown in FIG. 5. Young mice were used as acontrol group.

As a result of verifying H&E staining on the skin tissue of the back ofthe aged mice, it is general that the aging causes the progression ofhair loss and the thinning of the dermis layer. It can be confirmedthat, as for the young mice, the dermis layer is thick and a lot of hairfollicles, that is, where hairs grow, exist in the dermis layer(Y-control). Whereas, it can be confirmed that, as for the aged mice,the dermis layer become thin and the number of hair follicles issignificantly reduced (0-control). It could be verified that, when theaged mice were continuously immunized with an antigen (O-PspA) and arecombinant protein (0-FlaB-PspA), the dermis layer became thickened andthe number of hair follicles was increased. Particularly, it could beverified that the number of hair follicles was significantly increasedin the group immunized with FlaB-PspA recombinant protein.

As can be seen from the results of example 4, the histological assayconfirmed that the continuous immunization with antigens improved themorphological findings of the aged mice (results of example 2). It couldbe confirmed through the histological assay that the amelioration of theprogression of hair decoloration and hair loss of the aged mice, shownin the results of example 2, is due to the fact that the hair folliclesare maintained in the aged mice due to the continuous immunization. Inview of the results, it can be seen that the continuous immunizationwith the recombinant protein slows or prevents the progression of hairdecoloration and hair loss occurring due to aging.

Example 5: Change in Bone Marrow Cells in Bone Marrow ThroughImmunization with Recombinant Protein

The change in bone marrow cells in the bone marrow of the aged micethrough the continuous immunization was verified, and the results areshown in FIG. 6. Non-immunized aged mice were used as a control group.

As a result of observing the change in bone marrow cells with respect tocells separated from the bone marrow of the aged mice, there is nodifference between groups in view of cellularity.

As a result of confirming the proportions of bone marrow cells(megakaryocyte, myeloid, and erythroid lineages), it was verified thatthe bone marrow cells were generally well maintained and differentiatedin the aged mice.

In addition, as a result of verifying the frequency of hematopoieticstem cells through immuno-staining using CD34, which is a hematopoieticstem cell indicator, it could be verified that CD34 was increased in theaged mice continuously immunized with FlaB-PspA recombinant proteinrather than in the other groups.

As can be seen from the results of example 5, it could be verified thatthe continuous immunization with the recombinant protein is associatedwith not only the changes in the simple appearances but also theimmune-related effects in the old mice.

Example 6: Changes of Lymphatic System Organs Through Immunization withRecombinant Protein

After the continuous immunization for an appropriate period of time,organs were extracted from the mice of each group, and the morphologyand weight of each organ were measured. The measurement results areshown in FIG. 7. Young mice were used as a control group.

The tissues were extracted from the aged mice, and the morphologicalchange and weight of each of the organs were measured. As a result, thedifference caused by the continuous immunization was observed in thelymphatic system-related organs rather than in the other organs.Particularly, it was verified that the morphology and weight of theorgans in the group immunized with FlaB-PspA recombinant protein weresimilar to those of the young mice.

Thymic involution with age is a general phenomenon, but it was verifiedthat the thymus weight was increased in the aged mouse groupscontinuously immunized with antigens, and particularly, the thymusweight was significantly increased in the group immunized with FlaB-PspArecombinant protein. Besides, even though the mesenteric lymph nodes(MLNs) or the spleen, which are frequently used for systemicinflammation responses, mostly undergo a very hypertrophic morphologywith age due to the continuous infection, it was verified that thethymus weight was larger in the aged mice continuously immunized withFlaB-PspA recombinant protein rather than in the other groups; and thespleen weight was smaller in the aged mice continuously immunized withFlaB-PspA recombinant protein rather than in the other groups, and thespleen morphology of the aged mice continuously immunized with FlaB-PspArecombinant protein was also similar to that of the young mice.

As can be seen from the results of example 6, it could be verified thatthe aged mice continuously immunized with antigens exhibited improvedmorphological features of the immune-related organs. Particularly, theaged mice immunized with FlaB-PspA recombinant protein were observed tohave very similar organ morphological findings to the young mice.

Example 7: Change in Bone Mineral Density of Aged Mice ThroughImmunization with Recombinant Protein

Spines were extracted from the aged mice, and the change in bone mineraldensity through the immunization with the recombinant protein of theaged mice was measured using micro-computer tomography (microCT). Themeasurement results are shown in FIG. 8. Young mice were used as acontrol group.

After eight times of continuous immunization, the spines were extractedfrom the aged mice, and the change in bone mineral density throughimmunization was measured using microCT. As a result, it could beverified that the bone mineral density was significantly improved in thegroup immunized with the recombinant protein. In connection with example1, it could be thought that the facts that the body weight was notchanged depending on the number of times of immunization and the feedintake was increased in only the group immunized with the recombinantprotein are associated with the motion quantity of the aged mice, and itcould be explained that, in this regard, the bone mineral density wasmore significantly increased in the group continuously immunized withthe recombinant protein rather than in the other aged mouse groups.

Having described a preferred embodiment of the present invention, it isto be understood that variants and modifications thereof falling withinthe spirit of the invention may become apparent to those skilled in thisart, and the scope of this invention is to be determined by appendedclaims and their equivalents.

1-11. (canceled)
 12. A method for preventing, improving, or treatingmetabolic disease, the method comprising administering to a subject thecomposition comprising a recombinant protein of flagellin, which is theconstituent of Vibrio vulnificus flagella, fused with a pathogenicprotein antigen, as an active ingredient.
 13. The method according toclaim 12, wherein the flagellin is FlaB, which is flagellin of Vibriovulnificus.
 14. The method according to claim 12, wherein the pathogenicprotein antigen is surface protein A (PspA) of Streptococcus pneumonia.15. The method according to claim 12, wherein the recombinant protein isFlaB-PspA protein having an amino acid sequence of SEQ ID NO:
 1. 16. Themethod according to claim 12, wherein the recombinant protein enhancesimmunity.
 17. The method according to claim 12, wherein the metabolicdisease is caused by aging.
 18. The method according to claim 12,wherein the metabolic disease is a syndrome in which risk factors, suchas obesity, diabetes, hypertriglyceridemia, hypertension, cardiovasculardisease, and blood clotting, are shown together.